Increasing evidence suggests that there is an association between an inflammatory cascade and physiological shock [1, 2], diabetes [3, 4], cardiovascular diseases [5-10], acute cerebral stroke [11-15], Alzheimer's chronic disease [16], and various other diseases. This cascade is accompanied by activation of cells, expression of pro-inflammatory genes, down regulation of anti-inflammatory genes, attachment of leukocytes to the endothelium, elevated permeability of the endothelium, thrombosis, mast cell degranulation, apoptosis, growth factor release, and other events [17]. This evidence has opened up great opportunities in medicine to develop a variety of new anti-inflammatory interventions in an increasing number of diseases. Recent research designed to determine the origin of the trigger mechanisms of the inflammatory cascade in shock, multi-organ failure, and other diseases show that there exists an enhanced level of degradative enzymes that are targeted towards a variety of autologous proteins, protein structures, lipids, and lipid structures [1, 18-20]. This enzyme activity is not blocked to the same level as in non-disease control samples.
In shock, for example, digestive enzymes (e.g. chymotrypsin, trypsin, elastase, lipase, nuclease) synthesized in the pancreas find entry into the wall of the intestine [1]. Physiological shock is a life-threatening cardiovascular complication with high mortality that occurs in situations associated with trauma, including burns, surgery, ischemia, and sepsis. These traumas cause a reduced blood flow in the intestine, which in turn triggers an increased epithelial and endothelial permeability. This allows pancreatic enzymes to enter systemic circulation via the portal vein and/or intestinal lymphatics, where they produce a chain of events, including the production of inflammatory mediators (toxic protein fragments), inflammation, self-digestion of tissues, multi-organ failure, and eventually death. These enzymes have the ability to degrade almost all biological tissues and molecules, and, when exposed to the body during shock, lead to auto-digestion of matrix proteins and tissue cells in the intestinal wall and to the production of inflammatory mediators, which in turn further enhances the level of inflammation. Detection of these proteases in the blood can therefore diagnose a patient for the early stages of physiological shock, as well as for chronic and acute inflammation. It is also believed that the detection of lipases, amylases, and nucleases may be important for diagnosing these diseases. Furthermore the detection of proteases, lipases, amylases, and nucleases may also be important for many other diseases, including heart disease and cancer i.e., pancreatic cancer in particular.
As an additional example, diabetes is a disease characterized by excessive blood glucose levels [21]. Too much glucose in the blood can cause acute complications such as hypoglycemia, ketoacidosis and nonketotic hyperosmolar coma, as well as chronic complications such as cardiovascular disease, chronic renal failure, retinal damage (potentially resulting in blindness), several types of nerve damage, and microvascular damage (which can lead to impotence and poor healing). Glucose uptake from the blood is stimulated by the hormone insulin. Diabetes occurs when this hormone can't be synthesized by the body (type I) or when the body has resistance or decreased sensitivity to it (type II). For the latter case, recent evidence has shown that one particular pathogenesis of this insulin resistance may be proteolytic cleavage of the extracellular α-subunit of the insulin receptor by matrix metalloproteases (MMPs) [22]. It was shown, that spontaneously hypertensive rats (SHR) had significantly elevated MMP-9 protein levels in SHR microvessels, and elevated levels of leukocytes compared to normotensive Wistar-Kyoto rats. Furthermore, in-vivo micro-zymography showed enhanced cleavage by MMP-1,9 that co-localized with MMP-9 and was blocked by metal chelation. Using an antibody against the extracellular domain of the insulin receptor, this study further showed reduced density of the insulin receptor-α and a corresponding elevation of glucose and glycated hemoglobin in the blood, compared to the normotensive control. Treating the SHR with a broad spectrum MMP inhibitor, doxycycline, reversed all of these aforementioned trends. In other studies by Lee [23] and by Derosa [24], it was shown that there were elevated MMP-2, 9 levels in diabetic patients versus healthy patients. Together, all of these results show that one or more MMPs may be responsible for cleavage of the insulin receptor-alpha and corresponding insulin resistance, which in turn leads to type-2 diabetes. Detecting these matrix metalloproteases can therefore diagnose a developing insulin resistance during the early stages of type 2 diabetes.
Previous protease detection substrates and devices have included Fluorogenic Substrates [27, 28], Chromogenic Substrates [25, 26], FRET-Based Substrates [29, 30], EnzChek Assays, Immunohistochemical Assays, Fluorescence Polarization [31, 32] and Zymography [33]. These are protease assays based on cleavage of specific short amino-acid sequences designed to detect specific protease activity. The existing protease detection kits are based on 96 well plates with relatively large sample size (0.1 to 0.3 ml) each and they are not generally designed to detect cleavage of specific proteases. In general, these substrates are FRET type peptides which when cleaved by the protease produce an enhanced fluorescent signal or change in the fluorescent emission wavelength. Fluorescent PepTag® substrates are separated by gel electrophoresis after hydrolysis [34, 35]. These assays do not allow the detection of proteases directly in blood, cannot be separated easily from blood and plasma components, and are not useful for clinical diagnostics.
Other devices, systems and substrates for the detection of protein kinases and proteases have been designed [36, 37], however they appear not to be useful for rapid clinical diagnostic applications for the reasons discussed above.
Since the discovery of the link between the inflammatory cascade and physiological shock, diabetes, and potentially numerous other diseases, there have not been any detection platforms developed that could perform multiplex measurements of the clinical levels of disease related enzymes directly in blood or plasma. Therefore, there is a clear need for the rapid and quantitative detection of key disease related enzymes in formats that utilize minimal sample size and sample preparation. The present invention addresses these and other needs in the art.